Thermo Fisher Scientific
MitoTracker® Red CMXRos
M-7512
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The cell-permeant MitoTracker® contains a mildly thiol-reactive chloromethyl moiety and is used in live cells for labeling mitochondria. Additionally, some MitoTracker® probes are retained in the mitochondria after fixation. We are always on the lookout for fluorescent conjugates to stain cellular components in different primary cell lines and immortalized cells. In this case, we used MitoTracker® to stain the isolated mitochondria and track their biodistribution in live cells before fixation, using different concentrations of the dye.
Immunofluorescence
Human lung epithelial cells A549 (ATCC® CCL-185™) were cultured at 37ºC and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Invitrogen) and 2 mM L-Glutamine (Gibco). For experiments, cells were placed in wells of 24-well tissue culture plates containing round glass cover slips (12 mm diameter) and stained with Mitotracker (stock solution 1 mM) diluted 1:2000 or 1:20.000 for 45 min at 37ºC. Cells were washed with PBS and fixed with cold paraformaldehyde (3.2% in PBS) for 20 min at room temperature. After washing, cover slips were mounted on glass slides with mounting medium. Preparations were sealed and stored in the dark at 4°C.
None (Mitotracker dyes are already made with a fluorescent conjugate)
NA
Not necessary
Images were obtained with a Nikon A1R confocal scanning laser microscope. For MitoTracker Red, a 561 nm excitation, 570-620 nm emission filter was used.
When we studied the mitochondrial tree in human cells by fluorescence staining, we observed an excellent mitochondria staining with very good signal and low background at 1:2000 dilution. At 1:20.000 dilution, the signal was very low, but, in our hands, this staining results also in a very good signal after modification of the laser parameters (increasing laser intensity). Image. A) Mitotracker dilution 1:2.000.
The fixation method should be selected on the basis of the experimental requirements. Fixing cultured cells in 3.2% paraformaldehyde after the experiments results in excellent mitochondria staining with Mitotracker. Storage of Mitotracker: Always protected from light at -20°C (stock solution).
Quality products; easy to use; better than alternatives (í.e. plasmid transfection).
Needs a little optimization
Very good staining and reproducibility